The high-producing strain С3029/pGro7/pERDerp3 for the house dust mite allergen Der p 3 from Dermatophagoides pteronyssinus , expressing the recombinant protein in Escherichia coli in the soluble form, was constructed. A procedure was developed for the purification of the recombinant allergen. Crystals of the recombinant protein Der p 3 suitable for X-ray diffraction analysis were grown by the vapor-diffusion method. The X-ray diffraction data set was collected to 2.25 Å resolution at the European Synchrotron Radiation Facility (ESRF, France, ID23-1 beamline) at 100 K. The crystals belong to sp. gr. С 121 and contain two enzyme molecules per asymmetric unit.

Wild-type L-asparaginase from Wolinella succinogenes and its double mutant with V23Q and K24T substitutions were studied by molecular dynamics simulations. The molecular dynamics trajectories were analyzed. It was shown that the mobility of atoms in the mutant protein is much higher than that in the wild-type protein. The structural elements of the protein, the mobility of which is most strongly influenced by mutations, were revealed. The obtained data provide an explanation of changes in the substrate specificity of the mutant L-asparaginase from Wolinella succinogenes.

The three-dimensional structure of recombinant thermophilic ribokinase from Thermus speсies 2.9 was determined at 2.4 Å resolution using crystals, which were grown in microgravity by the counter-diffusion method. The X-ray diffraction data were collected at the SPring-8 synchrotron facility (Japan). An adenosine diphosphate molecule is located in the active site of the enzyme, and its environment is described. The three-dimensional structure of Thermus species 2.9 ribokinase is shown to be very similar to the structures of other enzymes of this family.

Using a molecular dynamics method, the state of the dimeric thymidine phosphorylase molecule from Escherichia coli in a complex with noncompetitive enzyme inhibitor 3'-azidothymidine and phosphate ion was studied on a trajectory of 50 ns. Previously obtained atomic coordinates of a complex of thymidine phosphorylase with azidothymidine and sulfate at a resolution of 1.52 Å were used as a starting model. It was demonstrated that both subunits of a dimeric enzyme molecule function asynchronously in a given time interval; moreover, each subunit maintains an open conformation. It was found that the nature of ligand at the nucleoside center affects the binding strength of phosphate in the phosphate center. In a complex with an inhibitor, both ligands over the entire time interval remain bound to the enzyme, while the release of phosphate from the active center is observed when simulating the behavior of thymidine phosphorylase in the presence of phosphate and thymidine substrate. The stabilizing effect of azidothymidine on phosphate binding is consistent with the behavior of azidothymidine as a noncompetitive inhibitor of thymidine phosphorylase.

Crystals of phosphopantetheine adenylyltransferase from Mycobacterium tuberculosis (PPAT Mt ), which were grown using 2-methyl-2,4-pentanediol (MPD) or ammonium sulfate as the precipitant, belong to sp. grs. R 32 and Р 3 2 , respectively. Crystals of the enzyme containing the ligand in the active site were obtained by the cocrystallization of the enzyme with functional substrates only in the presence of MPD (sp. gr. R 32). In the presence of ammonium sulfate, the ligand was not bound in the active site, and the cocrystallization resulted only in crystals of the apo form (sp. gr. Р 3 2 ). The crystal-packing patterns of the enzyme molecules and the structure of the apo form of РРАТ Mt in two crystal structures are compared in order to explain the binding patterns of the ligand in different crystal modifications. In the crystal modification P 3 2 , the molecules are more closely packed compared to the crystal modification R 32, and intermolecular contacts restrict the access to the active site.

The apo form of the double mutant of Wolinella succinogenes L-asparaginase (WAS) with V23Q and K24T substitutions in the flexible N-terminal loop (WASm), which exhibits an order of magnitude lower glutaminase activity compared to the wild-type enzyme, was crystallized in two modifications (sp. grs. P22121 and P21). The three-dimensional structure in two modifications was determined at 1.5 and 1.7 Å resolution, respectively. The three-dimensional structures and the molecular packing modes of the enzyme in two crystal modifications (monoclinic, sp. gr. P21, and orthorhombic, sp. gr. P22121) are compared. Intermolecular contacts and solvent channels in both crystal lattices are described. The orthorhombic crystals have a closer packing compared to the monoclinic crystals and lower water content (36.95 and 44.53%, respectively). However, the active sites in both structures are solvent accessible.

The mutant form of Wolinella succinogenes L-asparaginase (WASm) contains two replacements, V23Q and K24T, in the N -terminal flexible loop, which restricts the active center. This form of the enzyme has an order of magnitude lower glutaminase activity compared to the original enzyme (WAS). The 3D structure of WASm has been determined for the apo-form and WASm complexes with L-aspartic and L-glutamic amino acids with a resolution of 1.70, 1.65, and 2.0 Å, respectively. The amino acid residues of the N -terminal flexible loop are only partially localized on electron density maps. In the corresponding complexes, all active centers of the tetrameric enzyme molecules are fully occupied with aspartic and glutamic acids. Their environment has been described, and their location in the original and mutant enzyme coincides. The state of the active centers in the studied molecules has been considered. It has been shown that the active centers in all subunits of the WASm apo-enzyme and WASm/Glu complex are in open conformation, while those in three subunits of the WASm/Asp complex are closed and the conformation is open only in one subunit. The comparison of three-dimensional structures of the original and mutant enzyme complexes suggests that the decrease in glutaminase activity of WASm is caused by the increase in the flexibility of the residues in the N ‑terminal loop, which complicates the formation of the catalytically active closed form of the active center after the binding to the less specific substrate (glutamine).

Models of E. coli thymidine phosphorylase in complexes with the substrates — the complex with phosphate and the complex with phosphate and thymidine — were obtained by molecular docking calculations. The influence of the substrates on domain movements in the dimeric thymidine phosphorylase molecule was probed by molecular dynamics simulations. The two subunits were shown to function asynchronously. In the thymidine phosphorylase/phosphate and thymidine phosphorylase/phosphate/thymidine complexes, phosphate is more weakly bound in the active site and moves away from the phosphate-binding site during the 60-ns trajectory, whereas thymidine remains in the active site but undergoes conformational changes.

The double mutant of Wolinella succinogenes L-asparaginase (Was72) with the V23Q and K24T substitutions in the C-terminal region of the N-terminal loop enclosing the active site was crystallized in the apo form and in complexes with L-aspartic and L-glutamic acids. This mutant exhibits glutaminase activity eight times lower compared to the wild-type enzyme. Crystals of the apo enzyme were grown in two modifications (sp. gr. P 2 1 and sp. gr. P 22 1 2 1 ). Crystals grown in the presence of both aspartic and glutamic acids belong to the same space group ( P 2 1 ) but have different unit cell parameters. The X-ray diffraction data sets were collected from all types of crystals at 1.65–2.00 Å resolution. All X-ray diffraction data sets are suitable for the determination of the three-dimensional structure of the enzyme.

Recombinant purine nucleoside phosphorylase from the thermophilic Thermus thermophilus strain encoded by the TT_C0194 gene was purified to homogeneity. The crystallization conditions for the enzyme were found by the vapor-diffusion technique. The crystals of the enzyme suitable for X-ray diffraction were grown under microgravity conditions by the capillary counter-diffusion method. The crystals belong to sp. gr. P 2 1 2 1 2 1 and have the following unit-cell parameters: a = 89.9 Å, b = 121.0 Å, c = 215.7 Å, α = β = γ = 90°. The X-ray diffraction data set suitable for the determination of the three-dimensional structure of purine nucleoside phosphorylase was collected from the grown crystals at the SPring-8 synchrotron facility to 2.5 Å resolution.